Fibroblasts to hepatocytes: A nonstop flight into cell therapy for liver diseases?
Authors: Valerie Gouon‐Evans, Romina Fiorotto
Journal: Hepatology (2022)

Organ transplantation is still the sole curative treatment available for end‐stage liver diseases. However, the imbalance of high demand for liver organs and shortage of donor livers has driven the search for alternative regenerative therapy approaches, among which is hepatocyte cell transplantation.1 Hepatocytes for transplantation could be obtained from several sources, including organs that are currently discarded for surgical or other reasons and from alternative sources such as reprogramming of adult somatic cells, which brings the potential of autologous transplantation. The discovery of the induced pluripotent stem cell (iPSC) technology in 2006 by Takanashi and Yamanaka2 has led to extensive research in the field of regenerative medicine and has propelled the reprogramming of somatic cells for clinical applications. Scientists in the field of hepatology have applied this concept to the generation of hepatocyte‐like cells (CiHeps) or induced hepatocytes (iHeps).3 The iHeps can be generated from somatic cells (i.e., fibroblast, blood cells) either by generating an iPSC line followed by a stepwise differentiation protocol guided by the different stages of liver development or by direct reprogramming. In the case of direct reprogramming, fibroblasts, for example, can be cell‐fate converted into iHeps without the need for transitioning through an intermediate pluripotent state. Rather, cells transition to iHeps by repression of the fibroblast somatic state while inducing the hepatic program. Such a strategy relies on the activation of a mesenchymal to epithelial program and induction of expression of defined hepatic transcription factors. Direct differentiation is usually achieved by using viral vectors or culturing the cells with defined chemicals cocktails or more often by combining both approaches.4 As a result, a direct reprogramming process is generally less time and labor intensive than iPSC reprogramming and therefore more amenable to be translated into clinical applications. However, several benchmarks need to be achieved before iHep cell therapy can be applied to the clinic. The most challenging among these are safety, high efficiency and functionality of the cells, and tolerability when in the host. Despite these challenges, there has been substantial progress in advancing this technology with the goal of exploiting cell therapy as a curative treatment for chronic liver diseases. For instance, the study by Bai et al.,5 in this issue of Hepatology, represents an important proof of concept in the field of liver cell reprograming. In the study, a small molecule cocktail in the culture media was shown to directly reprogram mouse embryonic and neonatal skin fibroblasts into functional iHeps with the capability to repopulate mouse livers in vivo (Figure 1). Direct differentiation of mouse fibroblasts into iHeps has been achieved by other groups as recently reviewed by Rombaut et al.6; however, this is the first time that this could be achieved exclusively with a chemical approach. As part of their protocol, the authors first tested a combination of chemicals that can maintain primary hepatocytes in culture in an undifferentiated state for long‐term yet preserving their morphology. Next, they isolated fibroblasts from embryos of a fluorescent reporter mouse under control of the albumin promoter. By screening and combining previously described cocktails,7 they stepwise titrated the expression of master transcription factors involved in hepatocyte development and differentiation and monitored the in vitro conversion of fibroblasts into CiHeps via expression of albumin. Interestingly, the authors showed that the addition of vitamin C during the last part of the process increased the induction efficiency from 1%‐2% to 15%. Although the efficiency of this protocol is not superior to previously published ones,6 it displays a number of benefits. For example, the complete absence of viral vectors, which has not been achieved previously, decreases the risk of activation of oncogenes and therefore represents a safety advantage. Another strength of the study is the achieved functionality of these cells. Indeed, the authors provided a comprehensive characterization showing that CiHeps had transcriptomic, functional, and metabolic profiles that closely resemble those of mouse primary hepatocytes while losing mesenchymal features of origin. The investigators also reported the benefits of CiHeps in vivo. When injected intravenously in a concanavalin A–induced liver failure model, CiHeps protected mice from the liver damage as shown by increased survival (50%), improved liver serology, and decreased parenchymal necrosis. Although the authors failed to show whether CiHeps engrafted in the liver of these mice, calling into question the mechanism by which CiHeps are protective in 50% of transplanted mice, in a second model of liver injury (fumarylacetoacetate hydrolase‐deficient mouse model), the authors showed that CiHeps were able to engraft and repopulate up to 60% of the liver tissue in two out of six mice.FIGURE 1: Direct chemical induction of hepatocyte‐like cells (CiHeps) from mouse embryonic fibroblasts (MEFs) or neonatal skin fibroblasts (NSFs) with capacity for liver repopulation: MEFs or NSFs are chemically induced to generate up to 15% of chemically induced CiHeps along with chemically induced gastrointestinallike cells (CiGICs) and chemically induced keratinocytelike cells (CiKrts) as identified by single cell RNA sequencing analyses and whose crosstalk supports CiHep development. Hepatocyte nuclear factor 4 alpha (Hnf4a), forkhead box q1 (Foxq1), and kruppel‐like factor 4 (Klf4) were defined as key transcription factor modules for each chemically induced cell based on regulon activities. Importantly, transplantation of CiHeps rescues concanavalin A (Con a)‐induced liver failure in 50% of mice; and CiHeps are able to engraft in the liver in one of three FRG mice with 60% of efficiency repopulation.To shed light on the mechanism of transdifferentiation of fibroblasts, the authors then used a fluorescent lineage tracing system under the collagen type I alpha 2 promoter to label the fibroblasts during the transdifferentiation process. They found that iHeps originated directly from fibroblasts because there was no evidence, at least at the gene expression level, of a stem cell intermediate or developmental progenitor state during the process. The authors also observed a gradual and stepwise activation of hepatic transcription factors throughout the process (i.e., hepatocyte nuclear factor 4 alpha [Hnf4a], nuclear receptor subfamily 1 group i member 2, CCAAT enhancer binding protein alpha, nuclear receptor subfamily 1 group H member 4, hepatocyte nuclear factor 1 alpha, and forkhead box protein A 1‐3 [Foxa1‐3]) with HNF4a as the essential regulator of the hepatic reprogramming in the later stage of their protocol. This is consistent with the pioneer study from Sekiya et al.8 that provided the first demonstration that Hnf4a, in association with Foxa1, Foxa2, or Foxa3, can directly convert mouse embryonic and adult fibroblasts into CiHeps in vitro. In addition, the authors performed single‐cell RNA sequencing‐seq analysis of the cells derived from the induction of fibroblasts. That analysis identified five distinctive populations in their transdifferentiated cell pool, which were defined based on the differentially expressed gene as fibroblasts (1 and 2), CiHeps, cells with gastrointestinal features, and keratinocytelike cells. Of note, no other hepatic lineages (i.e., hepatoblasts and cholangiocytes) were detected. Whether these five populations relate to potential transcriptional heterogeneity of the starting population remains to be explored. These findings raise the question as to whether the other cell populations are perhaps playing a role in the differentiation and maintenance of CiHep phenotype. Indeed, when CiHeps were purified from and cultured in the absence of the other cell populations, the authors found that the transcriptomic profile of CiHeps was completely altered, suggesting that the other cell populations present in the pool are likely influencing the state and the differentiation of CiHeps. These findings open new questions, and further studies should investigate the potential crosstalk among these lineages in the hepatocyte differentiation process. Moreover, because all in vivo experiments in this study have been performed with unpurified cells, it is still unclear how the presence of other cell lineages could have impacted the outcome. Additional questions remain regarding the conversion efficiency in terms of future clinical applications, as it is well known that adult fibroblasts are less prone to conversion than embryonic fibroblasts and that translation to a human setting can be challenging. In summary, the study by Bai et al. clearly advances the field by demonstrating that direct chemical reprogramming can be used to safely generate functional CiHeps and that these cells are able to survive and expand in a diseased liver. These findings open future opportunities to be explored in translational applications and bring us closer to the promise of iHep‐based regenerative medicine in liver diseases.